TY - JOUR
T1 - Sequencing the hypervariable regions of human mitochondrial DNA using massively parallel sequencing
T2 - Enhanced data acquisition for DNA samples encountered in forensic testing
AU - Davis, Carey
AU - Peters, Dixie
AU - Warshauer, David
AU - King, Jonathan
AU - Budowle, Bruce
N1 - Publisher Copyright:
© 2014 Elsevier Ireland Ltd.
PY - 2015/3/1
Y1 - 2015/3/1
N2 - Mitochondrial DNA testing is a useful tool in the analysis of forensic biological evidence. In cases where nuclear DNA is damaged or limited in quantity, the higher copy number of mitochondrial genomes available in a sample can provide information about the source of a sample. Currently, Sanger-type sequencing (STS) is the primary method to develop mitochondrial DNA profiles. This method is laborious and time consuming. Massively parallel sequencing (MPS) can increase the amount of information obtained from mitochondrial DNA samples while improving turnaround time by decreasing the numbers of manipulations and more so by exploiting high throughput analyses to obtain interpretable results. In this study 18 buccal swabs, three different tissue samples from five individuals, and four bones samples from casework were sequenced at hypervariable regions I and II using STS and MPS. Sample enrichment for STS and MPS was PCR-based. Library preparation for MPS was performed using Nextera® XT DNA Sample Preparation Kit and sequencing was performed on the MiSeq™ (Illumina, Inc.). MPS yielded full concordance of base calls with STS results, and the newer methodology was able to resolve length heteroplasmy in homopolymeric regions. This study demonstrates short amplicon MPS of mitochondrial DNA is feasible, can provide information not possible with STS, and lays the groundwork for development of a whole genome sequencing strategy for degraded samples.
AB - Mitochondrial DNA testing is a useful tool in the analysis of forensic biological evidence. In cases where nuclear DNA is damaged or limited in quantity, the higher copy number of mitochondrial genomes available in a sample can provide information about the source of a sample. Currently, Sanger-type sequencing (STS) is the primary method to develop mitochondrial DNA profiles. This method is laborious and time consuming. Massively parallel sequencing (MPS) can increase the amount of information obtained from mitochondrial DNA samples while improving turnaround time by decreasing the numbers of manipulations and more so by exploiting high throughput analyses to obtain interpretable results. In this study 18 buccal swabs, three different tissue samples from five individuals, and four bones samples from casework were sequenced at hypervariable regions I and II using STS and MPS. Sample enrichment for STS and MPS was PCR-based. Library preparation for MPS was performed using Nextera® XT DNA Sample Preparation Kit and sequencing was performed on the MiSeq™ (Illumina, Inc.). MPS yielded full concordance of base calls with STS results, and the newer methodology was able to resolve length heteroplasmy in homopolymeric regions. This study demonstrates short amplicon MPS of mitochondrial DNA is feasible, can provide information not possible with STS, and lays the groundwork for development of a whole genome sequencing strategy for degraded samples.
KW - HVI
KW - HVII
KW - Heteroplasmy
KW - Massively parallel sequencing
KW - Mitochondrial DNA
KW - Sanger type sequencing
UR - http://www.scopus.com/inward/record.url?scp=84925374947&partnerID=8YFLogxK
U2 - 10.1016/j.legalmed.2014.10.004
DO - 10.1016/j.legalmed.2014.10.004
M3 - Article
C2 - 25459369
AN - SCOPUS:84925374947
SN - 1344-6223
VL - 17
SP - 123
EP - 127
JO - Legal Medicine
JF - Legal Medicine
IS - 2
ER -